This R21 responds to PA-03-145 "Ubiquitin and Ubiquitin-Iike Modifications Regulating Disease Processes". Rationale. The epithelial sodium channel (ENaC) affects plasma volume and blood pressure (BP) through sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Mutations in PY domains of ENaC, through which the ubiquitin ligase NEDD4L mediates channel degradation, lead to the hypertensive disorder, Liddle syndrome. Of two paralogous genes, NEDD4 and NEDD4L, NEDD4L has been identified recently as the more specific regulator of ENaC. NEDD4L was thought to lack a C2 apical targeting domain. We have found that: (I) mammalian NEDD4L directs the formation of multiple transcriptional isoforms that include or not a C2 domain; (2) a previously unidentified exon I generates a C2(+) isoform that is the most abundant in human kidney; (3) a common variant (A for G, 35%) generates a transcript that can only encode C2(-) isoforms; (4) in addition to genetic linkage, this polymorphism is associated with postural change in systolic BP in treated hypertensives, and with 24-hour BP monitoring and response to sodium loading in untreated hypertensives in the context of another variant affecting basolateral Na+,K+ ATPase. Hypothesis. With these data, our hypothesis is that the relative level of expression of NEDD4L isoforms, by impacting on apical ENaC density in ASDN, is an important regulator of sodium homeostasis and blood pressure. Research Aims. We propose to develop experimental animal models that will afford ultimately to investigate the impact of these two classes of NEDD4L isoforms on sodium balance and blood pressure in intact animals through gene targeting by homologous recombination in the mouse. Our aims are: (1) To generate two lines of animals that constitutively overexpress a C2(+) and a C2(-) isoform of NEDD41. in the ASDN using the Cre/Lox system for tissue specific activation; (2) To generate a line lacking expression of all NEDD4L isoforms in ASDN both as a test of the global significance of NEDD4L in nephron function and as a background to isolate through crossing the functional effect of the two isoforms overexpressed in Aim 1; (3) To perform a preliminary evaluation of the impact of these genetic manipulations on renal fund ion and blood pressure in response to challenges of sodium balance. Perspective. These animal models will afford in-depth evaluation of the contribution of the ubiquitin ligase NEDD4L. in ENaC regulation and analysis of genetic mechanisms of common forms of human essential hypertension.